GSM7118818: DF68555 500 nM VTP scATAC Seq; Homo sapiens; ATAC-seq
Frozen patient derived AML BM cells stored in liquid nitrogen were retrieved right before sample processing. To maximize the cellular viability recovery, samples were processed in batches according to thawing protocol. Briefly, cells were gently thawed in water bath at 37 °C for 2 minutes until partially thawed and immediately transferred to a 50 mL conical tube with 1ml of prewarmed media (RPMI1640 supplemented with 10% FBS) and centrifuged (300 g for 5 min). Cells were sequentially diluted 1:1 3 times with prewarmed RPMI containing 10% FBS. Cells were centrifuged at 300 x g for 5 minutes in a swinging bucket centrifuge. After finishing centrifugation, supernatant was disposed, the cell pellet was carefully re-suspended in 1 mL of 2% FBS/PBS, centrifuged at 300 x g for 5 minutes and resuspended in 900 uL of 2% FBS/PBS. To removed dead cells and debris, cells were passed through a 40 micron Flow-mi strainer and the cell concentration was determined with a Countess counting device. Nuclei were isolated following the demonstrated protocol for Nuclei isolation for single cell ATAC from 10X Genomics. Briefly, cells were lysed in chilled lysis buffer for 3 minutes, washed 3 times in chilled nuclei Wash Buffer, centrifuged for 5 minutes to remove the wash buffer and resuspened in Diluted Nuclei buffer (10X Genomics). The desired number of targeted nuclei (5000-10,000) in diluted Nuclei buffer were separated on the 10X Chromium Controller. Single cell ATAC-Seq libraries were generated following the manufacturer's protocol provided by 10X Genomics for sc-ATAC seq analysis.